1. Field of the Invention
Applicants' invention relates to the field of tumor growth regulation. More particularly, Applicants' invention concerns unique non-mammalian peptide hormone analogs of non-mammalian gonadotropin releasing hormone (GnRH) and the method for use of these analogs in the regulation of cell growth, particularly cancer cell growth.
2. Background Information
Gonadotropin-releasing hormone (GnRH) is a hormone known to be produced in the hypothalamus with binding affinity for the pituitary gland. When hypothalamic GnRH binds to the pituitary it causes the pituitary gland to release the gonadotropins (i.e. gonad stimulating hormones) luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Each of these pituitary hormones has a different effect depending on one's sex. One important effect is the production and secretion of the gonadal steroids estrogen and progestogen in sexually mature females and testosterone in males.
Before the chemical characterization of the mammalian GnRH it was realized that a hypothalamic substance regulated the production of pituitary LH and FSH (1) and that these gonadotropins regulated gonadal steroidogenesis. The delineation of mammalian GnRH enabled Applicants to synthesize this decapeptide and administer it systemically to humans (2). It was then recognized that a long acting superagonist of this mammalian GnRH effected a flare release of pituitary gonadotropins followed by their inhibition (3). The inhibition was effected by a down-regulation of the pituitary GnRH receptor which corresponded testosterone. This is a form of chemical castration.
Since certain types of tumors, such as certain breast (4) and prostate cancers (5), are now known to be dependent on gonadal steroids, mammalian GnRH analogs have been used to suppress gonadal steroids via their chemical castration activity (3). Thus, we know that the use of mammalian GnRH analogs is feasible as a treatment of certain cancers.
It was only with the development of sensitive and specific radioimmunoassays for GnRH and GnRH-like molecules that a very surprising finding was reported. That finding was initially described by Applicants. Applicants reported that GnRH-like molecules exist and function not only in the hypothalamic-pituitary axis, which functions as an endocrine system to distribute hormone systemically, but GnRH-like molecules also exist in extra-hypothalamic tissues (6-9) to provide a paracrine action i.e. localized signal secretion. It is now realized that paracrine action of GnRH-like substances have functions in the placenta, gonad, breast, prostate, etc., (10-13) as well as in many cancerous tumors (14-31). Even with this general knowledge, the effective use of mammalian GnRH analogs to act directly on particularly tumor tissue has not resulted. One of the goals of the present invention was to utilize novel forms of GnRH not previously envisioned for cancer therapy that bind to the tumor GnRH-like receptor with 50 to 1000 fold the activity of mammalian GnRH or its superagonist and have potent bioactivity in inhibiting tumor cell growth.
The initial studies on GnRH activity in tumor tissues and the human placenta utilized mammalian GnRH and its analogs, in accordance with the teaching that the human encodes for only one isoform of GnRH (32,33). In Applicants' studies in the human placenta Applicants localized and quantified the concentration of GnRH produced by the human placenta throughout pregnancy (34,35). Applicants demonstrated the synthesis and activity of a GnRH-like molecule in regulating human chorionic gonadotropin (hCG) and steroid production (36-38), and the release of a GnRH-like molecule into the maternal circulation (34). Applicants also demonstrated that high doses of mammalian GnRH could stimulate hCG and prostanoid production in a specific receptor mediated event and that GnRH-like production and activity in the human placenta is regulated by feedback interactions of estrogens and progesterone production (39-41). Thus, Applicants described and established the first paracrine system in human or mammalian physiology (42-45). Concomitantly, Dubois et al. (12) described a second paracrine system from the presence of somatostatin in the pancreas. Thereafter, Applicants and other investigators reported actions of mammalian GnRH on placental function and identified feedback interactions including activin, inhibitin, follistatin, neurotransmitters, prostaglandins, and steroids (46-63).
Using in situ localization a message to mammalian GnRH has been localized at the syncytio- and cytotrophoblast, as well as the stroma of the placenta (64-66). A gene for mammalian GnRH differing from hypothalamic GnRH by the inclusion of the first intron and a very long first exon has been reported (67-69). Multiple transcription sites have been identified for the GnRH gene in the placenta as well as in other reproductive tissues (70-72). Steroid regulatory sites on the promoter have also been identified (73,74). The functionality of the promoter is supported by the demonstration that mRNA for GnRH can be regulated by steroids (75-78).
Placental GnRH receptor activity and a GnRH mRNA have also been identified (79,80). The receptor number is highest in early gestation and down-regulated by 12-20 weeks, and still detectable in term placenta, although the mRNA for mammalian GnRH is not (79,80). This pattern of receptor activity parallels that of chorionic GnRH-like activity (6,34) and supports the hypothesis that chorionic GnRH may down-regulate its receptor, as does mammalian GnRH and its analogs at the pituitary level. Studies of Szilagyi et al. (81) and Currie et al. (82) have indicated down-regulation of chorionic receptors by mammalian GnRH analogs. In addition, estradiol has been shown to upregulate the placental GnRH receptor. Thus, there is substantial data to indicate a functional, regulated GnRH receptor in extrahypothalamic tissues.
In total, these studies have firmly established the presence of a hypothalamic-pituitary-gonadal axis in extra-hypothalamic tissue. Presently many other hypothalamic-like activities, such as by corticotropin-releasing hormone (CRH), have now been defined in the placenta and other tissues as well. Such paracrine axis are known in the pancreas, thyroid, gut, bone, brain, ovary, endometrium, eye, etc.
Of particular interest to this invention are previous reports of the presence of GnRH-like substances and receptors in numerous cancer tissues and their cell lines (15,17,20,23,25,26,30,31,83,84). GnRH-like activity and its receptors have been identified in the breast, bronchial, ovarian, endometrial, prostate, gastrointestinal tumors. The function of a GnRH-like substance and its receptors in tumor tissues is supported by the demonstration that mammalian GnRH can stimulate hCG from human and animal tumors and can inhibit cell growth in vitro. These findings have led to numerous studies of the effects of mammalian GnRH analogs on the expression of GnRH receptors, cell signal transduction, apoptosis, and overall growth of tumor cell lines (14,16,18,19,21,22,29,85-89). The growth of tumors in vivo has also been studied with individual case reports of patients responsive to mammalian GnRH analogs (24,27,28,90-92).
However, some very problematic findings from previous studies in both the placenta and tumor tissue has led to skepticism about the true role of mammalian GnRH analogs in both tissues. The GnRH receptor affinity for GnRH in both the placenta and tumors is on the order of 10−5 to 10−6 M (84,93,94). The biological significance of such a weak affinity in light of much lower levels of endogenous GnRH-like activity must be questioned. In addition, Applicants have observed in human pregnancy studies, both in vitro and in vivo, that mammalian GnRH appears to act as a partial agonist not a true agonist of tumor GnRH (38,95). When receptors are available, mammalian GnRH acts as an agonist of tumor GnRH, but when tumor receptors are low or occupied, mammalian GnRH competes with the more potent tumor GnRH resulting in a partial agonist action. Furthermore, Applicants and others have observed that certain antibodies for mammalian GnRH reacted with chorionic GnRH with a different affinity (96-99). These findings led Applicants to propose that neither the extra-hypothalamic GnRH nor its receptor are identical to mammalian GnRH and its pituitary receptor (100,101).
Applicants have defined yet another difference in extra-hypothalamic GnRH, i.e., its metabolism. The metabolism of a hormone is as important for maintaining biologically active concentrations of that hormone, as that which stimulates the hormone's synthesis and release. For GnRH, in the non-pregnant human, both in the pituitary and in the circulation, the predominant enzymatic degradation is directed to the 5-6 peptide bond catalyzed by an endopeptidase. Thus, existing analogs of the mammalian GnRH each bear a D-amino acid substituted in the 6 position. However, Applicants have isolated and characterized the dominant enzyme that degrades GnRH in the placenta and it is a post-proline peptidase acting to cleave the proline-glycine peptide bond at the 9-10 position (102,103). Applicants have recently obtained similar data for the metabolism of GnRH in breast tumor cells. Thus, there appears to be cell specific metabolism of GnRH at the placenta and breast tumor cells which differs from that in blood and the pituitary.
Since it appeared as though there was a different form of GnRH at work at the placenta and breast tumor cells, various isoforms of GnRH were investigated. Different isoforms of GnRH have been identified in non-mammalian species, such as fish and ayes. The unique sequence of these GnRH are known. Chicken I, chicken II, salmon, catfish, dogfish, lamprey and more recently herring GnRH have also been reported (33,106). In lower vertebrates a number of GnRH isoforms can be expressed in the same species (32,33,76,78,105,107-116). In most cases, each decapeptide conserves the first four, the sixth and in every case, the last two amino acids in the GnRH molecule, but have varying amino acids in the fifth, seventh and/or eighth position. These modifications render the molecule unique, having only weak affinity for the mammalian pituitary receptor, although conversely mammalian GnRH is active in many lower vertebrate classes.
As mentioned, in certain lower vertebrates a number of GnRH isoforms are expressed in the same species. In amphibians, a chicken II GnRH receptor as well as a mammalian GnRH receptor has been reported. However, it was not until 1994, when Dellovade et al. (117) and King et al. (118) described chicken II GnRH in musk shew, mole and bat brain, that the existence of multiple isoforms of GnRH in a mammal was realized. Even then, it was still thought that modern placental mammalian species did not encode or express other than mammalian GnRH. Recently however, chicken II GnRH has been characterized in the tree shew (119), guinea pig, and primate brain (120) and their separate genes have been described (121,122). Only this year has the code for the chicken II GnRH receptor been identified in human tissues.
In contrast, Applicants have proposed and obtained substantial data to support the hypothesis that non-mammalian isoforms of GnRH and their specific receptors are expressed in extra-hypothalamic tissues and that the non-mammalian GnRH molecules are the true ligands for these receptors (123,124). Applicants have also proposed that these GnRH molecules have specific roles in regulating cell growth and cell death and are pivotal in regulating cell growth of GnRH responsive tumors by a direct receptor mediated action on these tumor cells.
It is believed that the non-mammalian GnRH isoforms and analogs of the present invention may act either as a superagonist at the tumor tissue leading to tissue receptor down-regulation, or as a pure antagonist of the endogenous isoform of GnRH in the tumor tissue, acting via the tumor tissue receptor. The down-regulation of the GnRH receptor or the antagonism of the endogenous isoform of GnRH will provide for a reduction in cell proliferation and/or induce apoptosis. The specific action of the non-mammalian GnRH analog will compete at the tumor cell GnRH receptor(s) with the endogenous isoform of GnRH effecting an antagonism or a superagonistic down-regulation of the receptor, leading to cell death and regression of the tumor and inhibition of metastasis. Thus, this agent may be used to reduce tumor growth. To date, no such non-mammalian GnRH analog has been designed which has stability and tumor tissue specificity.
To date, little if any data, has been reported in relation to non-mammalian GnRH activity on tumor tissues. Chicken I GnRH and Lamprey GnRH (18,86,125,126) have been studied and limited activity was found. Applicants have studied these isoforms of GnRH and have found no or limited binding activity in chorionic tissues. On the other hand, Applicants have demonstrated greatly enhanced binding and bioactivity of chicken II GnRH and salmon GnRH analogs as compared to mammalian GnRH or its analogs in both breast cancer cells and placental tissue. Thus, Applicants have obtained data to support the hypothesis that certain non-mammalian GnRH analogs have enhanced receptor and bioactivity for tumor tissues and this finding taken together with the understanding of the unique metabolism of GnRH isoforms in cell specific sites have formed the basis of Applicants invention, i.e., the utilization of stable, cell-active analogs of non-mammalian GnRH isoforms to regulate tumor cell growth and the treatment of cancer. In addition, Applicants postulate that due to similar amino acid structures, Herring GnRH, Dogfish GnRH, and Catfish GnRH as well as other GnRH isoforms and analogs with similar amino acid structure should exhibit the same or similar binding and bioactivity.